PROJECT SUMMARY/ABSTRACT Cellular APOBEC3 proteins are potent HIV-1 restriction factors that catalyze the lethal hypermutation of viral genomes. HIV-1 protein Vif deactivates this restriction process by targeting APOBEC3 proteins for ubiquitylation and proteasomal degradation. Reactivation of APOBEC3 innate immune response pathways is a promising HIV-1 therapeutic strategy. Consequently, there is a critical need to identify key regulatory and structural components important for HIV-1 Vif-mediated destruction of cellular APOBEC3 restriction factors. The long-term goal of this proposal is to establish integrative proteomic and structural technologies for determination of virus-host interactions, especially in the context of HIV evasion of innate immune pathways. The next step to attaining this goal, and overall objective of this proposal, is to identify protein components and structural features of the Vif-APOBEC3 pathway that can be manipulated to protect APOBEC3 proteins from Vif-induced degradation. This objective will be reached by testing the central hypothesis that host-host protein interactions of the ubiquitin-proteasome system can be modulated to reactivate APOBEC3 restriction against HIV-1. To test this hypothesis the following three specific aims will be pursued. (Aim1) Identify candidate regulators of Vif-dependent APOBEC3 degradation. This first aim will be carried out by in vivo cross-linking and affinity purification mass spectrometry methodologies in combination with protein-protein interaction mapping analyses. (Aim2) Identify structural features and subunits essential to APOBEC3-Vif- CRL5 complex stability and function. Structural models of APOBEC3-Vif-CRL5 complexes will be generated using the integrative modeling platform which will merge solved structures (from crystallography, electron microscopy, SAX, etc.), with distance constraints and protein interaction interface data collected from structural cross-linking mass spectrometry experiments. (Aim3) Interrupt Vif-APOBEC3 ubiquitylation and degradation pathways to reactivate APOBEC3 restriction against HIV-1 infection. This will be accomplished using CRISPR/Cas9 gene deletion or ?knock-in? methods that will introduce protein interaction mutations into immunological cells to test their restrictive potential against HIV infection. Successful completion of the proposed research is expected to expand understanding of the molecular details involved in HIV-1 infection and evasion of innate immunity, as well as host response to retroviral pathogens. This will be a significant contribution as it will highlight new targets for improved therapeutic strategies for treatment of HIV-1 infection.